There is a need in the art for spatiotemporal modulation of gene expression using targeted light to activate an antisense or RNA interference (RNAi) process within a cell. Fulfillment of such a need will have an important impact in functional genomics. This is particularly the case in transparent model organisms, such a zebrafish, c. elegans and drosophila. Irradiation can be specifically targeted visually to known anatomic landmarks (Dmochowski and Tang, BioTechniques 43(2):161, 2007). Irradiating selected organs or groups of cells within organs permits specific genes to be turned off at any time during the development of the organism and with cell-scale resolution, thereby permitting an in depth spatiotemporal understanding of the role of particular genes in the whole organism. Several photo-caging approaches have been employed to modify antisense and RNAi molecules to make them photoactivatable.
Investigators have used a hairpin design to control the activity of an antisense molecule, wherein the antisense molecule is covalently attached to a complementary sense molecule via a photocleavable linker. The sense and antisense molecules bind one another in a hairpin conformation, preventing the antisense molecule from binding to its target mRNA. Irradiation cleaves the linker and releases the antisense molecule, which dissociates from the complementary sense molecule and binds its target mRNA. Studies by Tang and Dmochowski in the zebrafish focused on negatively charged peptide nucleic acid (ncPNA) or phosphorothioate DNA (sDNA) as the antisense molecule, and 2′-OMe-RNA as the complementary sense molecule in the hairpin (Tang et al., J. Am. Chem. Soc. 129:11000, 2007). Their work in human leukemia cells employed both DNA and phosphorothioated DNA as both sense and antisense components (Tang et al., Nucleic Acids Res. 36(2):559, 2008). In contrast, Shestopalov et al. controlled gene expression in the zebrafish using a morpholino polymer to form both the antisense and sense components in the hairpin (Shestopalov et al., Nature Chem. Biol. 3(10):650, 2007). Hairpin approaches have also been described for siRNA (U.S. Patent Applications 2005/0059028 and 2005/0282203, the disclosure of which are incorporated by reference herein). An important limitation of the hairpin approach is the difficulty or significant synthetic chemistry expertise that is required to assemble the hairpin (Tang and Dmochowski, Nature Protocols 1(6):3041, 2006).
Other investigators have approached light control of gene expression by applying sterically encumbered photolabile blocking (i.e. caging) groups to control the activity of siRNA molecules. Caging groups have been applied to the backbone phosphates in both random (Shah et al., Angew. Chem. Int. Ed. 44:1328, 2005) and targeted approaches (Shah and Friedman, Oligonucleolides 17:35, 2007; Nguyen et al., Biochim. Biophys. Acta 1758:394, 2006). The problem with random caging was that either the inactivation or the activation was not complete (that is, fully inactive siRNA could not be entirely activated and vice versa). The targeted approach provided fully active siRNAs after irradiation, but suffered from substantial residual activity of the caged precursor. Others have caged an exocyclic base moiety at specific RNA bases to control siRNA, and noted instability on storage as a problem (Mikat and Heckel, RNA 13:2341, 2007).
Natural and unnatural nucleobase ‘antisense’ nucleobase polymers can interfere with specific gene expression when they are complementary to the target gene's ‘sense’ mRNA (Wagner, Nature 372:333, 1994; Crooke, Annu. Rev. Med. 55:61, 2004; Antisense Drug Technology: Principles, Strategies, and Applications. Second Edition. Edited by Stanley T. Crooke. CRC Press and Taylor & Francis Group, New York. 2007). Such antisense nucleobase polymers can reduce the stability and/or expression of the target mRNA by a variety of mechanisms including RNase H-mediated selective mRNA degradation, interference, and steric blockade of ribosomal machinery.
Therefore, there is a need in the art to the provide caged, non-stem loop RNA 1 duplex molecules having a photocleavable base moiety to effect light activated inhibition of gene expression. The present disclosure was made to address this need.